Milton H. Werner

Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex.

The solution structure of the specific complex between the high mobility group (HMG) domain of SRY (hSRY-HMG), the protein encoded by the human testis-determining gene, and its DNA target site in the promoter of the müllerian inhibitory substance gene has been determined by multidimensional NMR spectroscopy. hSRY-HMG has a twisted L shape that presents a concave surface (made up of three helices and the N- and C-terminal strands) to the DNA for sequence-specific recognition. Binding of hSRY-HMG to its specific target site occurs exclusively in the minor groove and induces a large conformational change in the DNA. The DNA in the complex has an overall 70 degrees-80 degrees bend and is helically unwound relative to classical A- and B-DNA. The structure of the complex reveals the origin of sequence-specific binding within the HMG-1/HMG-2 family and provides a framework for understanding the effects of point mutations that cause 46X,Y sex reversal at the atomic level.

Intercalation, DNA Kinking, and the Control of Transcription

Biological processes involved in the control and regulation of transcription are dependent on protein-induced distortions in DNA structure that enhance the recruitment of proteins to their specific DNA targets. This function is often accomplished by accessory factors that bind sequence specifically and locally bend or kink the DNA. The recent determination of the three-dimensional structures of several protein-DNA complexes, involving proteins that perform such architectural tasks, brings to light a common theme of side chain intercalation as a mechanism capable of driving the deformation of the DNA helix. The protein scaffolds orienting the intercalating side chain (or side chains) are structurally diverse, presently comprising four distinct topologies that can accomplish the same task. The intercalating side chain (or side chains), however, is exclusively hydrophobic. Intercalation can either kink or bend the DNA, unstacking one or more adjacent base pairs and locally unwinding the DNA over as much as a full turn of helix. Despite these distortions, the return to B-DNA helical parameters generally occurs within the adjacent half-turns of DNA.

THE SOLUTION STRUCTURE OF THE HUMAN ETS1-DNA COMPLEX REVEALS A NOVEL MODE OF BINDING AND TRUE SIDE CHAIN INTERCALATION

Abstract The solution structure of a 24.4 kDa specific complex of the DNA-binding domain (DBD) of the human ETS1 (hETSi) oncoprotein with a 17-mer DNA has been solved by NMR. The interaction of the hETSi DBD with DNA reveals a surprising twist on the general features of helix-turn-helix (HTH)-DNA interactions. Major groove recognition involves the C-terminal two thirds of the HTH recognition helix, while minorgroove recognition occurs via true intercalation of the side chain of Trp-28, which extends from the minor to the major groove. This results in a sharp kink of ∼60° and a widening of the minor groove over one-half turn of the DNA. The orientation of the HTH element of the hETSi DBD with respect to the major groove is significantly rotated relative to other HTH proteins. These observations establish the ETS family of DNA-binding proteins as a distinct family of HTH proteins.

Refolding proteins by gel filtration chromatography

We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS‐1 protein, bovine ribonucelase A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of miligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X‐ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.

Correction of the NMR structure of the ETS1/DNA complex

The ETS family of transcription factors consists of a group of proteins that share a highly conserved 85 amino acid DNA-binding domain (DBD). This family recognizes a consensus sequence rich in purine bases with a central GGAA motif. A comparison of the published three-dimensional structures of the DBD/DNA complexes of ETS1 by NMR [Werner et al. (1995) Cell, 83, 761–771] and the related Pu.1 by X-ray crystallography [Kodandapani et al. (1996) Nature, 380, 456–460] reveals an apparent discrepancy in which the protein domains bind with opposite polarity to their target sequences. This surprising and highly unlikely result prompted us to reexamine our NMR structure. Additional NMR experiments now reveal an error in the original interpretation of the spectra defining the orientation of the ETS1-DBD on DNA. It was originally reported that the ETS1-DBD bound to DNA with a bipartite motif involving major groove recognition via a helix–turn–helix element and minor groove recognition via protein side-chain intercalation. The presence of intercalation was deduced on the basis of numerous NOEs between several amino acids in the protein and a resonance at 12.33 ppm originally assigned to a DNA imino proton. New NMR experiments now conclusively demonstrate that this resonance, which is located within the DNA imino proton region of the spectrum, arises from the hydroxyl proton of Tyr86. Realization of this error necessitated reanalysis of the intermolecular NOEs. This revealed that the orientation of the ETS1-DBD in the complex is opposite to that originally reported and that a tryptophan residue does not intercalate into the DNA. The calculation of a new ensemble of structures based on the corrected data indicates that the structure of the ETS1-DBD/DNA complex is indeed similar to the X-ray structure of the Pu.1-DBD/DNA complex.

Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins

BACKGROUND Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction. RESULTS We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences. CONCLUSIONS Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.

Molecular determinants ofmammalian sex

Mammalian male sex determination is controlled by a complex hierarchyof gene regulatory proteins and hormones, which promote male gonadal development and regression of the female primordia. At the core of this pathway lies the SRY protein, the master developmental switch for testicular differentiation and hence, the male sex. The three-dimensional structure of the SRY-DNA complex suggests a model of developmental gene regulation in which proteins that alter DNA structure and promote the assembly of higher-order nucleoprotein complexes play an essential role in the timing of cell specialization events.

Stopping death cold

The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, suggests how different combinations of proteins in the same family might be utilized to control apoptosis.

Understanding SRY-Related 46X,Y Sex Reversal at the Atomic Level

There is considerable evidence that the Y chromosome encoded testis determining factor, known as SRY, constitutes the primary active binary switch which regulates the transcription of a cascade of genes which in turn direct the development of the primordial gonad into the male testes (Goodfellow and Lovell-Badge 1993; McElreavey et al. 1993; Gustafson and Donahoe 1994; Haqq et al. 1994; Werner et al. 1996a). In the absence of functional SRY, either through mutations in SRY or because of chromosomal makeup (viz. XX females), the primordial gonad develops into ovaries. One potential target for SRY is the promoter for the gene encoding the Mullerian inhibtory substance (MIS) whose product is responsible for the regression of the female Mullerian ducts.